Invasive Prenatal Diagnosis

Introduction
Once a woman has been given a high risk of aneuploidy based on a Down screening, a structural anomaly detected on scan or because of her previous history an obstetrician would normally counsel the women on the options of invasive karyotyping.
This counselling should be based on the risk of aneuploidy, the voluntary nature of the test, the option of no testing, the technique of the proposed test, the procedure-related loss rate and other common complications associated with the test, the timing of the result and the possible management options depending on the result of the test. This decision to balance the potential risk of the loss of an unaffected fetus against that of having an affected child is a very difficult and traumatic one and it is
important that the parents are not rushed into a premature decision.

1. Amniocentesis
Amniocentesis should be performed after 15 weeks when the uterus is an abdominal organ and the proportion of fluid needed to be removed (15–10 ml) is relatively small compared to the overall liquor volume at this gestation (150–250 ml).

Procedure: The procedure is performed under aseptic conditions under continuous ultrasound guidance. Best practice is for the operator to introduce a gauge 22–20 needle percutaneously while he or she is continuously scanning using the free hand. The needle is preferably introduced into a cord free pool of liquor avoiding the placenta. Once in place the inner stylet of the needle is withdrawn and an initial 2 ml of amniotic fluid is withdrawn by an assistant and discarded to avoid maternal contamination. Then a further 15–20 ml is removed using a 10–20 ml syringe.
A few operators use a needle guide attached to the transducer, but this has the disadvantage of being less flexible if the needle needs to be realigned.

There is clearly a learning curve with any invasive procedure. Studies have demonstrated the significance of operator experience in terms of both failed attempts and miscarriage. Amniocentesis is therefore not a routine procedure and it is recommended by the Royal College of Obstetricians
and Gynaecologists (RGOG) that it is only performed by adequately trained individuals with at least 50–100 supervised procedures and 50 procedures per annum to maintain their skills. In general only two needle insertions should be attempted and if these fail then the woman should be referred to a tertiary level fetal medicine unit for repeat attempts.
The miscarriage rate for amniocentesis is generally quoted as 1:100 (1%) .


Post- Procedure:

• Leakage of amniotic fluid vaginally following an amniocentesis is relatively common occurring in up to 2% but is almost always self-limiting and associated with a normal outcome. 
• Post-amniocentesis chorioamnionitis is also rare  However, if signs of chorioamnionitis should be apparent following a recent amniocentesis a repeat amniocentesis with gram staining and culture of the amniotic fluid should be under taken. If infection is confirmed immediate emptying of the uterine cavity is needed to prevent maternal septicaemia.

2. Cytogenetic analysis

Amniotic fluid will contain fetal skin, urogenital and pulmonary epithelial cells and cells from the extra-embryonic membranes. The cells are first concentrated by centrifuging and are cultured for     7- 10 days with the result of the karyotype being available in 14–15 days. 

Culture failure occurs in about 0.5% and can be minimized by taking an adequate volume of fluid and limiting the delay from the sample being taken and sent to the laboratory. There is also the possibility of in vitro mitotic abnormalities occurring as a result of the culture process and therefore multiple

cultures are performed for each amniocentesis. Only if a mosaicism or karyotypic anomaly occurs in more than one culture is it likely to be of clinical significance and will need further investigation. 

More recently chromosome-specific probes and fluorescence in situ hybridization (FISH) techniques have been developed to detect numerical aberrations in interphase as well as, non-dividing cells, eliminating the need for prolonged cell culture. Most cytogenetic laboratories will now offer rapid prenatal diagnosis of amniotic fluid using either, fluorescence-based probes for short-tandem-repeat

(STR) markers on chromosomes 21, 13 and 18 and polymerase chain reaction (PCR) amplification of these STRs, or FISH and fluorescence-labelled DNA probes for chromosomes 21, 18 and 13 . This allows identification of the three commonest chromosomal anomalies within 3 working days.

3. Chorionic villus sampling (CVS)

CVS involves sampling of placental tissue rather than amniotic fluid and can be performed between 11 and 14 weeks. There are two routes used for CVS either transabdominal which is now the preferred option or the transcervical route if the former is not possible.

 There are various techniques for performing CVS and as there are no studies comparing techniques, the operators should use one that they are most familiar with. Nevertheless, the technique preferred

by theauthor is as follows. 

Procedure: It is an aseptic single operator technique, under continuous ultrasound guidance where the operator performs the ultrasound scan and the needling. A 20-gauge needle is inserted percutaneously

directly into the placenta being careful not to enter the amniotic sac. The stylet of the needle is removed by an assistant and a 20-ml syringe with 5 ml of heparinized saline is attached to the needle. Negative pressure is then applied to the syringe and the needle moved backwards and forwards in the placenta a few times to suction placental tissue within the needle. While the negative pressure is

maintained the needle is then withdrawn and the contents syringed into a sterile container.

Precautions: It is now recommended that CVS is not performed before 10 weeks because of the reported association of early CVS and isolated fetal limb disruption and oromandibular hypoplasia.

4. Fetal blood sampling

The availability of rapid karyotypic diagnosis with both CVS and amniocentesis and their considerably easier utility has resulted in fetal blood sampling (FBS) now rarely being used to assess karyotype. It is now almost exclusively used for the assessment of fetal anemia or infection.

Procedure: It is performed under sterile conditions with continuous ultrasound guidance. Again a single operator usually performs the ultrasound and needle insertion. A 20-gauge needle is inserted into the placental cord insertion or intra fetally into the intrahepatic vein, with colour Doppler mapping of the vessels. 

Risks: There are significantly greater risks of FBS than with amniocentesis or CVS. These include

• fetal bradycardia, 
• haemorrhage, 
• cord haematoma or tamponade and
• fetal death. 

Sampling from the intrahepatic cord appears to be safer than the placental cord insertion or free loops of cord. There needs to be immediate access to laboratory facilities for analysis of the fetal blood. Because of the significantly higher density of nucleated cells in an FBS a full karyotype culture of lymphocytes should be available within 48 hours.